Control of RNA degradation is a critical cell function, and gene expression is often regulated at the level of RNA stability. See, e.g., Shaw, G. and Kamen, R., Cell, 46:659-667 (1986). Nevertheless, relatively little is known about the bio-chemical pathways that mediate RNA degradation in mammalian systems. For instance, most if not all of the ribonucleases responsible for mRNA turnover in mammalian cells remain unidentified. This was reviewed in Brawerman, G., Cell, 57:9-10 (1989). Presently, the 2-5A system is believed to be the only well-characterized RNA degradation pathway from higher animals including man. See FIG. 1. See also, e.g., Kerr, I. M. and Brown, R. E., Proc. Natl. Acad. Sci. U.S.A., 75:256-260 (1978) and Cayley, P. J. et al., Biochem. Biophys Res. Commun., 108:1243-1250 (1982); reviewed in Sen, G. C. and Lengyel, P., J. Biol. Chem., 267:5017-5020 (1992). The activity of the 2-5A system is believed to be mediated by an endoribonuclease known as 2-5A-dependent RNase. See Clemens, M. J. and Williams, B. R. G., Cell, 13:565-572 (1978). 2-5A-dependent RNase is a unique enzyme in that it requires 2-5A, unusual oligoadenylates with 2', 5' phosphodiester linkages, p.sub.n (A2'p).sub.n A, for ribonuclease activity. See Kerr, I. M. and Brown, R. E., Proc. Natl. Acad. Sci. U.S.A., 75:256-260 (1978). 2-5A is produced from ATP by a family of synthetases in reactions requiring double-stranded RNA (dsRNA). See FIG. 1. See also Hovanessian, A. G. et al., Nature, 268:537-539 (1977); Marie, I. and Hovanessian, A. G., J. Biol. Chem., 267:9933-9939 (1992). 2-5A is unstable in cells and in cell-free systems due to the combined action of 2', 5'-phosphodiesterase and 5'-phosphatase. See Williams, B. R. G. et al.; Eur. J. Biochem., 92:455-562 (1978); and Johnson, M. I. and Hearl, W. G., J. Biol. Chem., 262:8377-8382 (1987). The interaction of 2-5A-dependent RNase and 2-5A(k.sub.d =4.times.10.sup.-11 M), Silverman, R. H. et al., Biol. Chem., 263:7336-7341 (1988), is highly specific. See Knight, M. et al., Nature, 288:189-192 (1980). 2-5A-dependent RNase is believed to have no detectable RNase activity until it is converted to its active state by binding to 2-5A. See Silverman, R. H., Anal. Biochem., 144:450-460 (1985). Activated 2-5A-dependent RNase cleaves single-stranded regions of RNA 3' of UpNp, with preference for UU and UA sequences. See Wreschner, D. H. et al., Nature, 289:414-417 (1981a); and Floyd-Smith, G. et al., Science, 212:1020-1032 (1981). Analysis of inactive 2-5A-dependent RNase from mouse liver revealed it to be a single polypeptide of approximately 80 kDa. See Silverman, R. H. et al., Biol. Chem., 263:7336-7341 (1988).
Although the full scope and biological significance of the 2-5A system remains unknown, studies on the molecular mechanisms of interferon action have provided at least some of the functions. Interferons .alpha., .beta. or .gamma. are believed to induce the accumulation of both 2-5A-dependent RNase, Jacobsen, H. et al., Virology, 125:496-501 (1983A) and Floyd-Smith, G., J. Cellular Biochem., 38:12-21 (1988), and 2-5A synthetases, Hovanessian, A. G. et al., Nature, 268:537-539 (1977), reviewed in Sen, G. C. and Lengyel, P., J. Biol. Chem., 267:5017-5020 (1992). Furthermore, several investigations have implicated the 2-5A system in the mechanism by which interferon inhibits the replication of picornaviruses. Indeed, 2-5A per se and highly specific 2-5A mediated rRNA cleavage products were induced in interferon-treated, encephalomyocarditis virus (EMCV)-infected cells. See Williams, B. R. G., Nature, 282:582-586 (1979); Wreschner, D. H. et al., Nucleic Acids Res., 9:1571-1581 (1981b); and Silverman, R. H. et al., Eur. J. Biochem., 124:131-138 (1982a). In addition, expression of 2-5A synthetase cDNA inhibited the replication of picornaviruses, Chebath, J., Nature, 330:587-588 (1987) and Rysiecki, E. F. et al., J. Interferon Res., 9:649-657 (1989), and the introduction of a 2-5A analogue inhibitor of 2-5A-dependent RNase into cells reduced the interferon-mediated inhibition of EMCV replication. See Watling, D. et al., EMBO J., 4:431-436 (1985). Further, 2-5A-dependent RNase levels were correlated with the anti-EMCV activity of interferon, Kumar, R. et al., J. Virol., 62:3175-3181 (1988), and EMCV-derived dsRNA both bound to and activated 2-5A synthetase in interferon-treated, infected cells. See Gribaudo, G. et al., J. Virol., 65:1948-1757 (1991).
The 2-5A system, however, almost certainly provides functions beyond the antipicornavirus activity of interferons. For instance, introduction of 2-5A into cells, Hovanessian, A. G. and Wood, J. N., Virology, 101:81-90 (1980), or expression of 2-5A synthetase cDNA, Rysiecki, G. et al., J. Interferon Res., 9:649-657 (1989), inhibits cell growth rates. Moreover, 2-5A-dependent RNase levels are elevated in growth arrested cells, Jacobsen, H. et al., Proc. Natl. Acad. Sci. U.S.A., 80:4954-4958 (1983b), and 2-5A synthetase, Stark, G. et al., Nature, 278:471-473 (1979), and 2-5A-dependent RNase levels are induced during cell differentiation. See, e.g., Krause, D. et al., Eur. J. Biochem., 146:611-618 (1985). Therefore, interesting correlations exist between 2-5A-dependent RNase and the fundamental control of cell growth and differentiation suggesting that the 2-5A system may function in general RNA metabolism. The ubiquitous presence of the 2-5A system in reptiles, avians and mammalians certainly supports a wider role for the pathway. See, for example, Cayley, P. J. et al., Biochem. Biophy. Res. Commun., 108:1243-1250 (1982).
Notwithstanding the importance of 2-5A-dependent RNase to the 2-5A system, 2-5A-dependent RNase enzymes having ribonuclease function have not been isolated, purified or sequenced heretofore. Consequently, there is a demand for isolated, active 2-5A-dependent RNases and their complete amino acid sequences, as well as a demand for encoding sequences for active 2-5A-dependent RNases.